Two distinct coagulase-dependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model

Abstract

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options.

Figure 1. Growth phenotypes of S. aureus Newman strains in different environments.

Growth phenotypes of S. aureus Newman strains under different growth conditions were analyzed 16 h after inoculation without agitation. Growth in RPMI 1640 leads to cluster formation of variable size (A). Growth in 3D-CoG also leads to cluster formation (B). Addition of 3 mg/ml fibrinogen to the medium (3D-CoG/Fib) resulted in the formation of discrete microcolonies of uniform diameter, surrounded by an inner pseudocapsule (yellow arrowheads) and an outer dense microcolony-associated meshwork MAM (blue arrowheads) (C). The sae mutant (Newman-29) formed clusters comparable to wildtype, even in 3D-CoG/Fib (D). A coa mutant formed microcolonies which were irregularly shaped in comparison to the wildtype, MAM formation was unaffected (E). A vWbp emp double mutant was unaffected in formation of the inner pseudocapsule but was devoid of any outer MAM (F). A1-F1: 40x oil immersion objective, scale bar 25 µm. A2-F2: 10x objective, scale bar 200 µm.

Figure 2. Microcolony and MAM diameter of S. aureus Newman strains after growth in 3D-CoG/Fib.

The average diameter of microcolonies and MAM was determined after 16 h of growth in 3D-CoG/Fib (A). Wildtype (wt), coa mutant and eap mutant formed microcolonies and MAM of comparable size. The vWbp emp double mutant did not form any MAM, despite being unaffected in pseudocapsule formation. A sae mutant (Newman-29) neither formed pseudocapsules nor MAM but grew in clusters significantly larger than wt colonies. USA300 microcolonies were comparable in size to wt, pseudocapsule formation was present, but the diameter of the MAM was significantly smaller compared to wt. Data are averaged from at least three independent experiments. Addition of plasmin (8 µg/ml) led to rapid degradation of both pseudocapsule and MAM surrounding S. aureus Newman colonies grown in 3D-CoG/Fib for 17h (B1–B4). The time stamp in the single panels is relative to plasmin addition, scale bar 25 µm.

Figure 3. Localization of Coa and Emp to S. aureus pseudocapsules.

Coa and Emp were detected in pseudocapsules by immunocytochemistry using rabbit antibodies specific for coagulase (anti-Coa, A) or Emp (anti-Emp, B and C). The pseudocapsule of the coa mutant is more irregularly shaped than its wildtype counterpart, illustrated by anti-Emp staining (C). Staphylococci were grown in 3D-CoG/Fib for 16 h and then fixed with 4% paraformaldehyde. Primary antibodies were detected by Alexa Fluor 555 anti-rabbit antibodies. Staphylococci were detected by staining DNA with DAPI (A1, B1, C1) and N-acetyl-glucosamine with FITC-Lectin (T. vulgaris) (A2, B2, B3). Staining of Coa and Emp was specific as the coa and vwbp emp mutant were not stained with the respective antisera (data not shown). Scale bar 7.5 µm. The images are representative of three independent experiments.

Figure 4. Presence of pseudocapsule and MAM structures in various clinical isolates.

Clinical S. aureus isolates were cultivated as described for strain Newman and evaluated after 16 h of growth. MSSA strain MP9-11 (A). MSSA strain MP3-11 (B). MSSA strain MP6-11 (C). MRSA strain MP10-11 (D). CA-MRSA strain USA300 (E). MSSA strain MP2-11 (F). MRSA strain ST239-CC8 (G). MSSA strain MP1-11 (H). Pseudocapsules (yellow arrowheads) and MAM-like structures (blue arrowheads) are indicated. A–C: scale bar 20 µm. D–H: scale bar 75 µm.

Figure 5. Degradation of fibrin-structures in later growth phases.

At later time points (here: 43 h after inoculation) single S. aureus Newman microcolonies (green arrowhead) started massive growth and dispersal by degradation of fibrin structures (A). Also comparatively small microcolonies are able to switch to this mode (blue arrowhead). A2 is a magnification of the inset in A1. An agr mutant is significantly less prone to degrade fibrin structures compared to the wildtype (B). The percentage of dispersing colonies was evaluated after 3 days by counting the respective microcolonies in a set of n independent wells (containing between 60 and 120 microcolonies per counted area, wt n = 11, agr n = 12, p<0.0001). Similar observations were made with CA-MRSA strain USA300 (C). A time lapse video of the lower left area in C is shown in Video S2. Scale bar sizes: A 150 µm, B 75 µm, C 50 µm.

Figure 6. Schematical drawing of the setup.

Staphylococci are grown in 3D-CoG/Fib in wells for 16 h in the absence of neutrophils. Then the medium is removed, stainings (e. g. Sytox Blue) can be applied and the 3D-CoG is overlaid with a 300 µm thin native spleen slice. Neutrophils migrate from the tissue slice into the 3D-CoG where they interact with staphylococci. The bottom of the well is suitable for confocal microscopy.

Figure 7. S. aureus Newman microcolonies are protected from neutrophils by MAM.

Neutrophils approached Newman wildtype microcolonies grown in 3D-CoG and immediately started phagocytosis (A, picture 3 h after challenge with neutrophils). The sae mutant (Newman-29) grown in 3D-CoG/Fib was attacked in the same way (B, picture 3 h after challenge with neutrophils). Wildtype microcolonies grown in 3D-CoG/Fib were not approached by neutrophils within 3 h (C, D). This can be depicted more clearly by time projection (0–3 h) of single frames from Video S3 (E). In contrast to this, the vWbp emp double mutant was readily approached by neutrophils which were only held back by the pseudocapsule (F–H, see Video S5). This neutrophil-free halo surrounding the microcolonies was measured and revealed a correlation with the MAM (I). Green: GFP-neutrophils; Blue: Sytox Blue-stained DNA; White: confocal reflection microscopy showing collagen fibers. Scale bar 50 µm. The microcolony in H is illustrated by a dotted line. The images are representative of at least three independent experiments. Data in I are averaged from three independent experiments.

Figure 8. The pseudocapsule is an additional protective barrier.

The pseudocapsules of vWbp emp double mutant microcolonies were protective against direct invasion of neutrophils into the microcolony (A, single frame from Video S6, 5 h after neutrophil challenge). After punctual rupture of the pseudocapsule, phagocytosis is initiated (B, single frame from Video S6, 27 min after A). After pseudocapsule rupture, phagocytosis is a rapid event (C, single frame from Video S7, 5 h after neutrophil challenge). USA300 barriers fulfill similar functions (D, single frame from Video S15, 3.5–5 h after neutrophil challenge). Direct contact of staphylococci with neutrophils led to massive neutrophil cell lysis/necrosis, visualized by Sytox Blue staining (yellow arrowheads, E–H, single frames from Video S8, 5 h after neutrophil challenge). In E, the microcolony outline is illustrated by a dotted line. Green: GFP-neutrophils; Blue: Sytox Blue-stained DNA; White: confocal reflection microscopy showing collagen fibers. Scale bar 20 µm.

Figure 9. The thrombin inhibitor argatroban antagonizes staphylococcal barrier activity.

S. aureus Newman was grown in 3D-CoG/Fib in the presence of different argatroban concentrations. After 16 h of growth, the growth phenotypes were analyzed: at 10 nM argatroban, the MAM was diminished and at higher concentrations also pseudocapsule formation was absent (A). Challenging the system with neutrophils after 16–17 h revealed loss of both barrier functions in a concentration-dependent manner (B). Scale bar 25 µm. The images are representative of three independent experiments. Data are averaged from three independent experiments.

Figure 10. Proposed model for the differential actions of Coa and vWbp.

S. aureus Newman forms two concentric barriers in the presence of fibrinogen in the growth medium: the outer MAM and the inner pseudocapsule; both contain fibrin. The MAM is dependent on vWbp and inhibits neutrophil immigration into the vicinity of the microcolony. The pseudocapsule is partially dependent on Coa and acts as a second barrier preventing neutrophil invasion of the microcolony.